Project Summary Chronic infection by the Hepatitis B virus (HBV) is a leading cause of human cancer worldwide, and is strongly associated with development of cirrhosis and hepatocellular carcinoma (HCC). The 17-kDa HBx protein of HBV is a causative tumorigenic agent and affects multiple cellular processes, either on its own or together with the proteins it targets. Though the oncogenic potential of HBx has been demonstrated, neither its structure nor the molecular mechanisms by which it mediates liver-associated diseases are known. The major obstacles have been the sparing solubility, lack of significant homology to characterized proteins and intrinsic disorder. Our studies have succeeded in obtaining HBx in highly soluble forms and for the first time shown that HBx is an [Fe- S]-binding protein. Our long-term goal is to establish the chemical nature of the cofactor and its involvement in driving protein conformation and reactivities, ultimately translating this molecular and structural knowledge to HBxs? extended functional repertoire. Our central hypothesis is that the [Fe-S] cluster is a common feature of HBxs across all genotypes. We propose that the [Fe-S] cofactor confers structure in an otherwise disordered protein and modulates protein reactivity and interactions by (at least) three distinct pathways: a) protein-protein interactions, by changing the oligomeric or conformational status of HBx, b) redox mechanisms involving either i) electron transfer processes to cofactors of target proteins or ii) regulatory processes as a response to cellular redox status and generation of ROS, c) Fe- or [Fe-S]- transfer mechanisms, by which HBx can act as a scaffold for iron-trafficking to regulate iron homeostasis and downstream molecular pathways. Our specific aims will test these hypotheses by: (Aim 1) establishing the biologically relevant form of the cofactor, and if both observed [4Fe] (stable) and [2Fe] (transient) forms are physiologically relevant. We will identify the cluster ligands, cysteine residues likely involved in disulfides and examine how clinical mutations and large sequence deletions may affect the cofactor and thus HBx function. (Aim 2) Establish the type, location and effects of the [Fe-S] cluster on the protein structure (disorder-to-order transition) and whether cluster incorporation drives protein folding. If successful, this step will set the stage for solving by solution NMR methods the highly sought structure of HBx, either on its own or together with cellular binding partners. (Aim 3) Establish a link between the type and redox form of the [Fe-S] cofactor and HBx biological activity. The expected overall impact of this innovative proposal is that it will fundamentally advance our understanding of HBx on the molecular and structural level, which is currently missing. Because HBx is a potential target for the development of anti-cancer drugs, determining the role(s) of the [Fe-S] cofactor and the linked structure/function relationships, will glean its part in viral-induced pathogenesis and offer new therapeutic avenues.